The concept of using antisense oligonucleotides (ASOs) to reduce protein expression was first proposed by Zamecnik and Stephenson in 1978 when they demonstrated that an oligonucleotide complementary to 13 nucleotides of the Rous sarcoma virus 35S RNA inhibited virus production in Rous infected chick embryo fibroblasts (Zamecnik et al., Proc. Natl. Acad. Sci., 1978, 75, 280-284). Advances in antisense therapeutics since this time have been substantial, with the first therapeutic ASO being approved for human use in 1998 (Marwick, J. Am. Med. Assoc., 1998, 280, 871). The recent introduction of RNA interference as a method to analyze gene function in invertibrates and plants (Fraser et al., Nature, 2000, 408, 325-330) has suggested that double-stranded RNA, specifically small nucleotide interfering RNAs (siRNAs), may also have therapeutic applications (Vickers et al., J. Biol. Chem., 2003, 278, 7108-7118).
When double-stranded RNA molecules are introduced into cells they are metabolized to small 21-23 nucleotide siRNAs with two-nucleotide (2-nt) 3′-overhangs via the endogenous ribonuclease Dicer (Grishok et al., Science, 2000, 287, 2494-2497; and Zamore et al., Cell, 2000, 101, 25-33). Inside cells, siRNA molecules bind to an RNA-induced silencing protein complex. This complex, which possesses helicase activity, unwinds the double-stranded siRNA, thereby allowing the antisense strand to bind to the targeted RNA. An endonuclease then hydrolyzes the target RNA (Zamore et al., Cell, 2000, 101, 25-33; and Zamore, Science, 2002, 296, 1265-1269). Since ultimately a single stranded RNA molecule binds to the target RNA molecule according to Watson-Crick base pairing rules, siRNA driven RNA interference is essentially an antisense mechanism of action (Vickers et al., J. Biol. Chem., 2003, 278, 7108-7118). siRNA duplexes used for silencing mammalian genes in cultured cells are usually chemically synthesized 21-23 nucleotide (21-23-nt) siRNAs, where the siRNA's sense and antisense strands are paired, containing 2-nt 3′-overhangs (Harborth et al., J. Cell. Sci., 2001, 114, 4557-4565). siRNA molecules were designed with a 2 nucleotide (2 nt) 3′-overhang because this form of siRNA has been shown to be most effective in vitro (Elbashir et al., Nature, 2001, 411, 494-498). The 5′-hydroxyl is not blocked by methylation or a 5′-phosphodiester linkage, as both prevent the 5′-phosphorylation of the antisense siRNA, a step necessary for target RNA degradation inside cells (Nykanen et al., Cell, 2001, 107, 309-321; and Schwartz et al., Mol. Cell., 2002, 10, 537-548).
Zamore and others have reported that single-stranded antisense oligonucleotides are less potent and less effective than siRNAs at reducing gene transcript levels (Zamore et al., 2000, Cell, 101, 25-33; and Caplen et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 9742-9747). As the antisense molecules used in those studies were single-stranded unmodified RNA, which are rapidly degraded by endogenous nucleases, here we compare antisense siRNA molecules to “second generation” phosphorothioate (PS) oligodeoxynucleotides modified to contain 2′-O-methoxyethyls (MOEs), both in vitro and in vivo. These second generation antisense oligonucleotides are chimeric molecules, which by design, contain a stretch of RNAse H sensitive 2′deoxy residues in the middle, flanked on both sides with a region of 2′MOE modifications. These molecules, termed MOE gapmers, take advantage of: 1) 2′-MOE modifications, which form higher affinity complexes and possess higher nuclease resistance relative to “first generation” PS oligonucleotides, resulting in increased ASO potency both in vitro and in vivo (Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; Dean, N. M., Pharmacology of 2′-O-(2-methoxy)-ethyl modified antisense oligonucleotides, in Antisense Technology: Principles, Strategies and Applications, S. Crooke, Editor, Marcel Dekker, 2001; and Kurreck, Eur. J. Biochem., 2003, 270, 1628-1644); and 2) PS 2′deoxyoligonucleotides, which when duplexed with RNA, serve as efficient substrates for the robust endogenous RNAse H antisense-mediated cleavage of RNA (Baker et al., Biochim. Biophys. Acta, 1999, 1489, 3-18). Indeed, antisense MOE gapmer reduction of target mRNA levels can be in the order of 85-90% of control levels (Crooke et al., Annu. Rev. Pharmacol. Toxicol., 1996, 36, 107-129; and Baker et al., Biochim. Biophys. Acta, 1999, 1489, 3-18).
Antisense oligonucleotides are known to preferentially accumulate in hepatic tissue in vivo (Cossum et al., J. Pharmacol. Exp. Ther., 1993, 267, 1181-1190; and Graham et al., J. Pharmacol. Exp. Therap., 1998, 286, 447-458). Nestle and colleagues have previously reported that cultured hepatocytes rapidly internalize antisense compounds in the absence of cationic lipid transfection reagents (Nestle et al., J. Invest. Dermatol., 1994, 103, 569-575). These observations are likely related to the remarkable transport rates displayed by hepatocytes, where fluid-phase endocytosis at the basolateral membrane is estimated to be 8% of the total membrane surface area per minute per cell (Crawford, Semin. Liver Dis., 1996, 16, 169-189).
The present invention provides a primary hepatocyte cell model that demonstrates in vitro antisense oligonucleotide uptake and intracellular trafficking similar to postulated in vivo antisense oligonucleotide uptake and trafficking. In particular, the present invention demonstrates antisense oligonucleotide mediated target mRNA reduction in primary hepatocytes without cationic lipid carriers, analogous to that postulated to occur in vivo. The results described herein suggest that the mechanism of cellular uptake of single strand MOE gapmers and double strand siRNA are different. Single strand MOE gapmers, but likely not double strand siRNA, are taken up in hepatocytes in vivo and in vitro without aid of cationic lipids. When siRNA molecules are transfected into cells, they produce a dose dependent reduction of target gene expression.